The colon cultivates a vast community of beneficial microbes that generates a diverse mix of >500 small (50- 1500 Da) molecules. The mechanistic links between this microbial metabolome and human health remain poorly understood. A systematic effort, using a combination of approaches, is urgently needed to delineate the subset of metabolites that impact health and to understand how these bioactive molecules exert their systemic effects. We hypothesize that the intestinal epithelium selectively absorbs a limited number of luminal metabolites with bioactive potential while actively excluding potential toxins. This exploratory study seeks to address this hypothesis by first mapping the stratification of luminal metabolites across functional segments of the mouse colon using a discovery metabolomics approach that combines the dynamic range and precise identification of 1H NMR with the sensitivity and resolution of GC-MS (Aim-1). In addition, we will use a novel Ussing chamber approach to delineate the repertoire of fecal solutes that are selectively transported across the colonic epithelium, and the stratification of these transport processes across colon segments (Aim 2). This approach will also enable us to follow the fate of luminal solutes that are metabolically transformed during transit through the colonic epithelial cells. Our study will bring together two laboratories with extensive expertise in intestinal transport physiology (Lytle) and analytical chemistry (Larive) to discover novel pathways of communication between the gut microbiome and its host. Our partnership will yield new information on the stratification of luminal metabolites along the colon, on the capacity of each segment to absorb them, and on the concomitant biochemical transformation of fecal solutes during epithelial absorption. This work will serve as a springboard for an extended program of collaborative research aimed at understanding how microbial metabolites are transported and metabolized by the colonic epithelium.